The evolution of resistant pathogenic organisms has compromised the utility of many of the most valuable classes of antibiotics and necessitates the development of new agents to maintain our ability to combat infectious disease. Trimethoprim-sulfamethoxazole (TMP-SMX; Bactrim), discovered in the 1950s, is now one of the mainstay oral therapies for the treatment of community-acquired methicillin-resistant Staphylococcus aureus (MRSA), a clinically significant Gram-positive pathogen associated with skin and skin structure infections (SSSI). However, TMP-SMX is only effective against a narrow range of Gram-positive bacteria and does not cover other common pathogens associated with SSSI such as Streptococcus pyogenes. Additionally, there has been a steady increase in the number of TMP-SMX-resistant strains of MRSA. Both the narrow spectrum and much of the resistance are related to changes in the sequence of dihydrofolate reductase (DHFR), the target of TMP. We have been focused on the development of next-generation antifolates that are effective inhibitors of the naturally TMP-insensitive S. pyogenes as well as the wild-type and TMP-resistant MRSA. Using structure-based design, we have developed a class of antibiotics known as the propargyl-linked antifolates (PLAs) that show potent antibacterial activity against both pathogens, oral bioavailability, low levels of resistance and efficacy in a murine model of MRSA. In this proposal, we describe efforts to further refine this lead series to improve spectrum of coverage while optimizing key pharmacokinetic properties. These efforts are described in three specific aims. In the first aim, we assess the current clinical spectrum for the PLAs and study the molecular basis of TMP resistance to better inform compound optimization and selection. The second aim describes the design, synthesis and evaluation of superior analogs against insensitive and resistant forms of the target. In the final aim, select candidate compounds are evaluated in murine infection models to determine efficacy and pharmacokinetic parameters. Through this work, we anticipate identification of several promising candidate compounds that would be attractive for further translational development.